Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/76—Albumins
  • C07K14/765—Serum albumin, e.g. HSA
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
  • A61P11/06—Antiasthmatics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/10—Antimycotics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
  • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
  • A61P31/18—Antivirals for RNA viruses for HIV
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/20—Antivirals for DNA viruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/20—Antivirals for DNA viruses
  • A61P31/22—Antivirals for DNA viruses for herpes viruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/06—Antianaemics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
  • G01N2333/715—Assays involving receptors, cell surface antigens or cell surface determinants for cytokines; for lymphokines; for interferons
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• CXC chemokine receptor 4 (CXCR4) antagonistic polypeptide A CXC chemokine receptor 4 (CXCR4) antagonistic polypeptide
• the present invention relates to a CXC chemokine receptor 4 (CXCR4) antagonistic polypeptide (protein) that inhibits infection of CXCR4 depending viruses such as HIV-I and blocks tumor cell migration mediated by CXCL12 binding to CXCR4: Human circulating antiviral albumin fragment (ALB408-423) and its therapeutic and diagnostic use.
• CXCR4 CXC chemokine receptor 4
• the invention includes the naturally occurring form of ALB408- 423 and fragments derived therefrom and/or analogues or derivatives, and finally a medicament containing said natural, recombinant and synthetic peptides to be used for medicinal indications and to be used as a diagnostic agent.
• the invention includes modified forms and derivatives of ALB408-423 that have a particularly favorable therapeutic effectiveness.
• nucleic acid probe hybridizing to ALB408-423 or one of its fragments and/or derivatives, and antibodies or antagonists directed against ALB408-423 or one of its fragments and/or derivatives, for diagnostic or therapeutic purposes, especially in viral diseases for the treatment of HIV-I and HIV-2 infections as well as for the treatment of neoplastic diseases to prevent cancer cell metastasis or for the treatment of chronic inflammatory diseases such as asthma, pulmonary fibrosis or rheumatoid arthritis
• Chemokine receptors are expressed on the surface of certain cells, which interact with cytokines called chemokines.
• the CXC chemokine receptor 4 (CXCR4) is a G- protein-coupled receptor that transduces signals of its endogenous ligand, the chemokine CXCL12 (stromal cell-derived factor-1, SDF-I). Following interaction of CXCR4/CXCL12 intracellular calcium (Ca 2+ ) ions fluxes are triggered. This causes cellular responses, including chemotaxis allowing cells to travel within the organ- ism.
• CXCR4 is expressed on myeloid cells, T-lymphocytes, B-lymphocytes, epithelial cells, endothelial cells and dendritic cells.
• the chemokine CXCL12 is the only known agonistic ligand of CXCR4.
• the interaction between CXCL12 and CXCR4 plays a crucial role in the migration of progenitor cells during embryologic development of the cardiovascular, hemopoietic or central nervous systems. This interaction is also known to be involved in several diseases such as HIV infec- tion/AIDS, cancer cell metastasis, leukemia cell progression, pulmonary fibrosis and rheumatoid arthritis. It is assumed that this interaction may be a critical therapeutic target in all of these diseases. Substances interfering with CXCR4/CXCL12 signaling are assumed to have drug potential, e.g.
• receptor antagonists are ligands or drugs that do not induce a biological response, i.e. cell migration or Ca 2+ signaling, upon binding to their receptor. Receptor antagonists are useful drugs already in clinical use (e.g.
• angiotensin antagonists that can block HIV-I infection (CCR5 antagonist) or decrease agonist mediated cellular responses.
• CCR5 antagonist CCR5 antagonist
• Interaction of receptor antagonists with the receptor inhibits the function of an agonist.
• Most drug antagonists achieve their potency by competing with endogenous ligands or substrates at structurally defined binding sites on receptors.
• CXCR4 antagonists block cancer cell migration and hence metastasis.
• CXCR4 is expressed on the surface of a variety of cells (myeloid cells, T-lymphocytes, B-lymphocytes, epithelial cells, endothelial cells and dendritic cells) as well as in 23 different types of cancer cells.
• CXCL12-CXCR4 interaction is involved in metastasis of several types of cancer, including cancer of the breast, kidney, prostate, lung, and pancreas, and melanoma, neuroblastoma, non-Hodgkin's lymphoma, multiple myeloma, ovarian cancer, and malignant brain tumors (reviewed in Tsutsumi et al., 2007, Peptide Science 88: 279 - 289). It has been shown that CXCR4 antagonists such as T140 analogous suppress CXCL12 induced pancreatic cell migration and invasion or breast carcinoma cell migration in vitro and in vivo (reviewed in Tsutsumi et al., 2007, Peptide Science 88: 279 - 289).
• CXCR4 antagonists effectively suppress invasion and adhesion of small cell lung cancer (SCLC) in vitro (reviewed in Tsutsumi et al., 2007, Peptide Science 88: 279 - 289), confirming the involvement of the CXCL12-CXCR4 interaction in SCLC metastasis.
• CXCR4/CXCL12 interaction is also involved in the development of precursor-B (pre-B) acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL.
• pre-B precursor-B
• ALL acute lymphoblastic leukemia
• CLL chronic lymphocytic leukemia
• CXCR4 antagonists also attenuate migration of pre-B ALL cells (reviewed in Tsutsumi et al., 2007, Peptide Science 88: 279 - 289).
• rheumatoid arthritis is caused by CXCR4 expressing CD4+ memory T cell accumulation in the inflamed synovium.
• CXCL12 concentration in the synovium of rheumatoid arthritis patients are highly elevated thereby attract- ing memory T cell.
• CXCR4 antagonistic molecules block migration of memory T cells into the synovium (reviewed in Tsutsumi et al., 2007, Peptide Science 88: 279 - 289).
• CXCR4 antagonists do not only inhibit binding of the agonist CXCL12 to CXCR4 but also prevent interaction of the HIV glycoprotein gpl20 with CXCR4 thereby inhibiting virus infection.
• the human immunodeficiency viruses 1 and 2 use cell surface expressed CD4 as primary receptor and the chemokine receptors CCR5 or CXCR4 as coreceptors for cell entry.
• Viruses that infect cells via CD4 and CXCR4 are termed CXCR4 (X4) tropic, HIV-I variants using CD4 and CCR5 as R5 tropic, and those that can use both coreceptors as dualtropic.
• X4 tropic HIV-I variants can only be found in about 50% of all AIDS patients whereas R5 tropic HIV variants predominate in earlier stages and the asymptomatic phase of HIV-I infection. It has been shown that X4 tropic HIV-I infection can be blocked in vitro and in HIV-I infected humans by treating cells or patients with CXCR4 antagonists such as AMD3100. Interestingly, Maraviroc, a CCR5 antagonist, is the first clinical approved drug in AIDS therapy that blocks infection of R5 tropic HIV-I variants (reviewed by Tsibris and Kuritzkes, 2007, Annual Review of Medicine 58:445-459).
• chemokine receptor CXCR4 is an attractive therapeutic target for the treatment of HIV/AIDS, cancer associated pathologies and chronic inflammatory diseases like asthma or pulmonary fibrosis.
• CXCR4 antagonists blocking CXCL12 mediated cellular responses could inhibit these important pathways of disease development and progression. Summary of the invention
• the invention pertains to a peptide having the following amino acid sequence:
• Z represents number of from 0 to 10 amino acid residues.
• the peptide of the present invention is also related to the peptides of the inven- tion, in particular ALB408-423 peptide according the invention, wherein single or several amino acid residues in the sequence have been exchanged, deleted or added, or chemical modifications on single amino acids of the peptides of the invention, in particular ALB408-423 have been introduced which have similar or the same biological or pharmacological activity of the peptides of the invention, in particular ALB408-423.
• those peptides are concerned which can easily be obtained by exchanging amino acids of the sequence in a conservative manner which means to exchange hydrophobic amino acids against hydrophobic ones or aromatic against other aromatic amino acids or basic amino acids against other basic amino acids and the like. This is well known to skilled person.
• retro-inverso peptides of the peptides of the invention are in the scope of the present invention, as well as other derivatives stabilizing the peptide bond against peptidases.
• derivative means all length fragments including truncations at the N and C terminus, ALB408-423 containing amino acid residue substitutions including D- amino acid residues and modified amino acid residues as well as peptides containing disulfide bonds and extension at the N and C terminus.
• Another subject matter of the present invention are polynucleotides coding for the peptides of the invention, in particular ALB408-423 and/or its derivatives.
• the polynucleotides of the invention are characterized by being constituted of DNA, RNA, genomic DNA or PNA.
• Polynucleotides coding for the peptides of the invention shall be used for recombinant peptide expression in pro- or eukaryotic cells, mutagenesis studies, cloning in vectors of interest, in particular those that can be used for gene transfer approaches.
• a further subject of the present invention is a vector containing the polynucleotides according to the invention.
• Vectors encoding polynucleotide sequences coding for the peptides of the invention shall be used for recombinant peptide expression in pro- or eukaryotic cells, mutagenesis studies, and particular for ALB408-423 gene transfer into eukaryotic cells.
• a subject of the present invention is a genetically engineered host cell containing the vector according to the invention.
• a genetically engineered ALB408- 423 or related derivative expressing transgenic cell can be used for gene therapy approaches allowing expression and secretion of ALB408-423 and related derivatives in individuals being in the need of CXCR4 antagonsits, in particular cancer and AIDS patients.
• Yet another subject of the invention is an antibody directed against the polypep- tides according to the invention.
• Those antibodies are useful to detect ALB408-423 and related peptides in body samples like blood, serum, plasma in ELISA, RIA or immune fluorescence for diagnostic purposes.
• the peptides of the invention can be administered in a method for the treatment of patients in need of the peptides of the invention, in particular ALB408-423.
• a further subject of the present invention is a method for the treatment of patients in need of ALB408-423 inhibition by administering therapeutic amounts of an antagonist/inhibitor the peptides of the invention.
• ALB408-423 and its derivatives are CXCR4 antagonists allowing to treat several diseases such as HIV infec- tion/AIDS, cancer cell metastasis, leukemia cell progression, pulmonary fibrosis and rheumatoid arthritis and other cancer and inflammatory diseases.
• a galenic formulation consisting of polypeptides of the invention is also subject matter of the invention.
• the peptide of the invention can be provided by a process comprising an extraction from hemofiltrate by cation-exchange extraction followed by elution of adsorbed substances, renewed cation-exchange chromatography of the extract containing the peptides, and fractional reverse-phase chromatography.
• the process for the manufacturing of the peptides according to the invention can be performed by solid-phase synthesis in terms of a Merrifield synthesis or liquid-phase synthesis by methods known per se to the skilled person using protected amino acids, and its purification.
• a further process for the manufacturing of the peptides according to the invention employs methods of heterologous expression known to the skilled person using common biotechnological vectors.
• a subject of the present invention is a diagnostic agent containing a poly- or monoclonal antibody of the invention or containing the nucleic acid or mRNA coding for the peptides of the invention, in particular ALB408-423.
• the diagnostic agent of the invention contains the peptides, or polynucleotides of the invention for use in test systems for assaying levels of this substance in samples such as tissue, plasma, urine and cerebrospinal fluid.
• diagnostic agents and test systems detecting the peptides of the invention are used for assaying tissue, plasma, urine and cerebrospinal fluid levels of this substance by means of mass-spectrometric methods, such as MALDI-MS or ESI-MS, in connection with sample preparation by RP-HPLC, protein precipitation and/or solid-phase extraction.
• mass-spectrometric methods such as MALDI-MS or ESI-MS
• Subject of the invention is also a diagnostic agent containing the peptides of the invention as markers for viral diseases, bacterial and fungal infections, inflammatory and neoplastic processes, and as markers in inflammatory processes, disturbed inflammation reactions, tumor diseases, growth disorders, diseases of the immune system, and as markers in bone diseases.
• the present invention provides also a medicament containing the peptides of the invention as an active ingredient of galenic forms for oral, intravenous, intramuscular, intracutaneous, subcutaneous, intrathecal administration, and as an aerosol for transpulmonary administration.
• the peptides, the polynucleotides, the antibodies/antagonists, and the galenic formulation according to the invnetion can be used for the treatment of viral diseases, especially HIV-I, HIV-2, cytomegalovirus, herpes simplex virus (types 1 and 2), varicella zoster virus, hepatitis A and hepatitis B viruses, influenza virus, polio virus, rhinovirus, rubella virus, measles virus, rabies virus, Rous sarcoma virus, Epstein-Barr virus, and for the treatment of bacterial and fungal infections, inflammatory processes, disturbed inflammation reactions, tumor diseases, growth disorders, neuronal diseases, diseases of blood clotting and hematopoiesis, vascular diseases, diseases of the immune system, and for wound and bone healing.
• viral diseases especially HIV-I, HIV-2, cytomegalovirus, herpes simplex virus (types 1 and 2), varicella zoster virus, hepatitis A and hepati
• Figure 1 A-F show details of the isolation of ALB408-423 from human hemofiltrate.
• Figure 2 shows ALB408-423 containing fraction 31 blocks HIV-I NL4-3 infection.
• Figure 3 shows chemically synthesized ALB408-423 specifically blocks X4 tropic HIV-I infection.
• Figure 4 shows ALB408-423 blocks X4 tropic lentiviral infection.
• Figure 5 depicts antiviral activities of ALB derivatives.
• Figure 6 shows antiviral activities of ALB derivatives.
• Figure 7 shows antiviral activities of ALB derivatives.
• Figure 8 shows antiviral activities of ALB derivatives.
• Figure 9 shows cytotoxicity assay of ALB derivatives.
• Figure 10 shows ALB408-423 and truncated ALB derivatives specifically block X4 tropic HIV-I infection.
• Figure 11 shows ALB408-423 and derivatives inhibit X4 tropic HIV-I infection of peripheral blood mononuclear cells (PBMC).
• PBMC peripheral blood mononuclear cells
• Figure 12 shows ALB408-423 inhibits binding of CXCL12 to CXCR4.
• Figure 13 shows ALB408-423 is not a CXCR4, CCR5 or CXCRl agonist.
• Figure 14 shows ALB408-423 specifically inhibits CXCL12-evoked Ca 2+ mobilization in CXCR4 expressing cells.
• Figure 15 shows ALB408-423 blocks CXCL12 mediated CXCR4 internalization.
• Figure 16 shows ALB408-423 dose dependently blocks CXCL-12 mediated migra- tion of Jurkat T cells.
• Figure 17 shows CXCR4 antagonistic activities of ALB derivatives.
• ALB408-423 could be surprisingly isolated from human hemofiltrate by means of chromatographical methods and a biological assay.
• the biochemical characterization of the peptide according to the invention was effected by mass spectrometry including a complete sequence analysis of the amino acids.
• the peptide has the following amino acid sequence Seq ID No 8:
• the molecular weight of the peptide ALB408-423 according to the invention is:
• the isoelectric point (pi) of the peptide ALB408-423 according to the invention is 10.3.
• the peptide according to the invention is a fragment comprising 16 amino acids of the known human plasma protein serum albumin (Accession No. NP000468), which consists of 585 amino acids in its processed form.
• Human albumin is a soluble monomeric serum protein having a molecular weight of about 65,000 that accounts for more than half the total plasma protein (concentration : 3.5 to 5 g/dl).
• the function of human albumin is predominantly described as a carrier molecule for all kinds of hydrophobic as well as hydrophilic substances, e.g., steroid and peptide hormones, fatty acids, vitamins, pharmaceuticals and cations.
• Albumin Due to its very high serum concentration, it contributes substantially to the stabilization of the blood pH, the extracellular liquid volume and the maintenance of colloid-osmotic pressure.
• Albumin has a globular structure stabilized by a high number of disulfide bridges and is usually not glycosylated, but alterations due to acetylation, enzymatic glycosylation and non-enzymatic glycosylation occur frequently in the course of molecular ageing or upon pathophysiological changes.
• pre-pro-albumin having 609 amino acids; the N- terminal signal peptide comprising 18 amino acids is cleaved of intracellular ⁇ upon entry in the endoplasmic reticulum; another 6 amino acids is removed in the Golgi apparatus before the mature albumin comprising 585 amino acids is secreted by the liver cells.
• the clearance of albumin takes place through the kidney, the gastrointestinal tract and in the tissue cells of the liver.
• the peptide sequence of ALB408-423 according to the invention starts with amino acid 408 and thus comprises the amino acids 408 to 423 of the circulating form of albumin. It is evidently produced by natural processing of the albumin precursor by corresponding proteases.
• the peptide according to the invention is an antagonist for the CXC chemokine receptor 4 (CXCR4) and causes a suppression of HIV-I infection and replication in human cells as well as a suppression of CXCL12/CXCR4 induced cellular responses such as cell migration, Ca 2+ mobilization or CXCR4 internalization.
• CXCR4 CXC chemokine receptor 4
• the peptide according to the invention is obtainable by chromatographic purifica- tion from human hemofiltrate (HF).
• HF is obtained in large amounts during ultrafiltration of the blood of kidney patients (example 1).
• HF contains all peptides and proteins circulating in human blood with a molecular weight below 30 kDa.
• Peptides and proteins in HF were extracted using cation exchange chromatography. Column bound peptides and proteins were eluted with buffers systems of various pH values and eluates were subjected to reversed phase chromatography (example 1). To identify fractions blocking HIV-I infection, peptide fractions were dissolved in PBS and added to HIV permissive indicator cells.
• Albumin; (ALB)” encompassing amino acid residues 408-423 (ALB408-423) (example 2).
• ALB408-423 specifically blocks X4 tropic HIV-I variants but has no effect on R5 tropic HIV-I (example 4 and 5) infection in indicator cells.
• ALB408-423 also suppressed infection of X4 tropic HIV-2 (example 5).
• ALB408-423 derivatives such as ALB408-419 or ALB L408I-419 displaying increased antiviral activity compared to wild type ALB408-423 (example 6). None of the ALB derivatives is cytotoxic (example 7).
• ALB408-423, ALB408-419 and ALB L408I-419 dose dependently blocked infection of a variety of X4 tropic but not R5 tropic HIV-I variants in indicator cells (example 8) or primary blood mononuclear cells (example 9). All these data indicate a specific interaction of ALB408-423 or its derivatives with the HIV coreceptor CXCR4.
• ALB408-423 directly binds to and interacts with CXCR4 thereby preventing binding of CXCL12, the natural CXCR4 agonist (examples 10-12).
• ALB408-423 or its derivatives alone do not induce Ca 2+ mobilization via CXCR4 or other chemokines receptor such as CCR5 and CXCRl indicating that ALB408-423 is a CXCR4 antagonists per definition (examples 10-12 and 14).
• CXCL12 mediated cell migration (example 13) and CXCR4 receptor internalization could be blocked as well providing further evidence that ALB408-423 is a CXCR4 antagonist (example 12).
• ALB408-423 a human serum albumin fragment was identified by screening a HF derived peptide library using an HIV-I infection inhibition assay.
• the chemically synthesized peptide and derivatives thereof dose depen- dently block X4 tropic HIV-I and HIV-2 infection by a direct interaction with the
• ALB408-423 and its derivatives act antagonistically as they do not mediate cellular responses and suppress activity of CXCL12, the natural occurring CXCR4 agonist. These data are evidence that ALB408-423 is the first human CXCR4 antagonist. ALB408-423 and its derivatives might be useful in the treat- merit of individuals infected with X4 tropic HIV-I, to prevent cancer metastasis and to interfere with chronic inflammatory diseases where CXCR4/CXCL12 signaling is involved and suppresses CXCL12 mediated signaling through CXCR4.
• the peptide according to the invention as well as analogues, fragments and derivatives of the peptide, its cDNA, its gene and antibodies that neutralize the activity of ALB408-423 can be employed as medicaments. Its biological activity corresponds to that of virus-inhibiting, cancer cell migration inhibiting and CXCR4 antagonistic substances.
• ALB408-423 specifically binds CXCR4 thereby preventing infection of CXCR4 tropic HIV-I variants and binding of the natural CXCR4 agonist CXCL12.
• the peptide according to the invention can be administered in a way usual for peptides on a parenteral, intravenous, intramuscular, intranasal, local- topic, subcutaneous or buccal route.
• the amount of peptide to be administered is from 1 ⁇ g to 1 g per unit dose per day.
• the activity of the peptide according to the invention can be inhibited by administering appropriate inhibitors/antagonists.
• the diagnostic agent according to the invention contains poly- or monoclonal antibodies against the peptide according to the invention, optionally in a fluorescence-labeled or radioactively labeled form, to be employed in a per se known ELISA or RIA.
• the diagnostic agent according to the invention contains DNA, RNA and/or PNA, optionally in a modified and/or labeled form, for use in test systems known to the skilled person, such as PCR or fingerprinting.
• the diagnostic agent according to the invention consists of a mass-spectrometric method (MALDI or ESI-MS) that unequivocally detects the substance qualitatively and quantitatively from its singly of multiply charged ions (parent ions or product ions after MS-MS fragmentation) after a corresponding sample preparation and enrichment (separation of large proteins by precipitation, enrichment of ALB408- 423 by chromatography or RP media, solid-phase extraction).
• MALDI or ESI-MS mass-spectrometric method
• Example 1 Isolation of the antiviral Iy effective ALB408-423 from human hemofiltrate
• Human hemofiltrate is optionally diluted with water and acidified.
• the pH value is preferably from 1.5 to 3.5, especially from 2.5 to 3.0.
• the hemofiltrate is passed through a cation exchanger, for example, a support material modified with sulfonic acid groups (Fraktogel SP-650 (M), Merck, Darmstadt, Germany).
• the peptides bound to the cation exchanger are eluted with a relatively high concentration of a salt solution.
• the ionic strength of the eluate is about that of a 0.5 to 1 M ammonium acetate solution.
• the collected eluate is subjected to another cation exchange chromatography.
• This chromatography is preferably a fractional elution with buffers having increasing pH values.
• fractions containing the peptide according to the invention are further purified by preparative reverse-phase chromatography followed by semipreparative reverse-phase chromatography, for example, on C18-modified support materials.
• the degree of purification is preferably monitored using analytical reverse-phase chromatography, for example, on C18-modified support materials.
• Buffer A Hemofiltrate pH 2.7, conductivity 5.5 mS/cm
• Buffer B 0.5 M ammonium acetate
• Equipment Autopilot Chromatographic System (PerSeptive Biosystems, Wiesbaden, Germany)
• rinsing is effected with several column volumes of 5 mM HCI.
• the elution of the bound peptides is effected as a batch elution with 0.5 M ammonium acetate.
• a complete elution of the peptides is achieved through a ramping pH value (6.8 to 7.2) and ramping conductivity (56 mS/cm) in about 5 liters of eluate.
• ammonium acetate eluates of the batch extraction are combined in an amount of 10,000 liters of hemofiltrate peptide.
• the peptide extract is charged onto the preparative cation exchanger with the addition of completely desalted water having a conductivity of 5.5 mS/cm.
• the column After charging the raw extract over 240 min, the column is rinsed with 0.01 M HCI until the conductivity is below 1 mS/cm. Elution is performed in several steps with the buffers stated below.
• Eluates 1-7 are designated as pH pool I-VII. They are separately collected and finally rinsed with completely desalted water. Elution is effected until a new base line is reached, elution volumes of from 10 to 25 liters being reached for the individual pH pools I to VII.
• the individual pH pools are separated by reverse-phase chromatography for fractionating and simultaneous desalting.
• HIV inhibition test were performed by seeding 4000 P4-R5 MAGI cells (P. Charneau et al., J. MoI. Biol. 241 : 651, 1994) in 100 ⁇ l of DMEM (10% FCS, 100 U/ml penicillin G, and 100 ⁇ g/ml streptomycin sulfate). P4-R5 cells are stably trans- fected with an LTR-lacZ cassette and upon successful infection by HIV-I will express ⁇ -galactosidase in a Tat-dependent manner, which can be detected in a chemiluminescence test. On the following day, aliquots of the fractions were added.
• lyophilized fractions were resuspended in 80 ⁇ l of DMEM, and 25 ⁇ l each thereof was pipetted to P4-R5 cells, incubated at 37 0 C for 1 hour and subsequently infected with HIV-I NL4_3 (1 ng of p24 antigen).
• Virus stocks were obtained by transient infection of 293T cells with proviral DNA by the calcium phosphate method (CalPhosTM Mammalian Transfection Kit, Clontech). Virus stocks were harvested 48 hours post transfection, filtrated and used for infection. Three days post infection, ⁇ -galactosidase activity in infected P4-R5 cells was detected using GalScreen assay (Tropix) as recommended by the manufacturer.
• Buffer A 100% water
• Buffer B 80% acetonitrile, 20% water, 10 mM HCI
• Packing material Bakerbond RP-C18, 15-30 ⁇ m, 300 A) Buffer A: 30% methanol, 70% water, 10 mM HCI Buffer B: 100% methanol, 10 mM HCI Gradient: 0-15% B in 40 ml
• Bioactive fractions 5 + 6 from the previous chromatography were separated through an analytical reverse-phase column. Aliquots were tested in a bioassay (HIV inhibition assay). Fractions 51 to 57 contained the substance according to the invention ( Figure ID).
• Packing material RP-C4, 5 ⁇ m, 100 A, Biotek Silica, Ostringen, Germany
• Buffer A water, 0.1% TFA Buffer B: 80% acetonitrile, 20% water, 0.1% TFA Gradient: 0-5% B in 2 min, 5-35% B in 60 min, 35-100% B in 3 min Flow rate: 7 ml/min Detection: 214 nm and 280 nm Chromatographic equipment: Kontron Fractions: 1 min each from min 1 7th step: Analytical reverse-phase C18 chromatography:
• Bioactive fractions 51-57 from the previous chromatography were separated through an analytical reverse-phase column. Aliquots were tested in a bioassay. Fraction 31 contained the substance according to the invention in a pure form ( Figure IE).
• Packing material RP-C18, 5 ⁇ m, 300 A, Vydac (Hesperia, USA)
• Buffer A water, 0.1% TFA Buffer B: 80% acetonitrile, 20% water, 0.1% TFA
• the pure substance according to the invention was contained in fraction 31 and was then examined in a bioassay in a dose-dependent manner and characterized by peptide chemistry (example 2).
• the purified native peptide was analyzed by means of MS-MS coupling analysis (ESI-TRAP) supplied by the company PROTEOM EFACTORY AG, Dorotheenstr. 94, 10117 Berlin (Germany), by a data base comparison of the established ESI MS-MS masses by means of the Mascot search engine, which resulted in the following sequence with the highest probability:
• ESI-TRAP MS-MS coupling analysis
• a further data base comparison with the SwissProt data base shows that the peptide sequence has 100% identity with amino acids 408-423 of the human protein serum albumin (Accession No. NP000468), and the sequence contains the amino acids: LVRYTKKVPQVSTPTL.
• Purified fraction 31 is active in the HIV-I inhibition bioassav
• FIG. 2 ALB408-423 containing fraction 31 blocks HIV-I NL4-3 infection.
• TZM-bl cells were incubated with serial dilutions of fraction 31 and were then infected with X4 tropic HIV-1NL4-3. 3 days later infection rates were determined by GalScreen assay. Shown are mean values ⁇ standard deviation from triplicate infections relative to PBS treated controls (100%).
• ALB408-423 was performed by means of conventional solid-phase synthesis on a peptide synthesizer 9050 (Applied Biosystems) using the known Fmoc chemistry.
• the peptide obtained was purified by reverse-phase chromatography, and its identity and purity were established by analytical RP- HPLC and by the MALDI-MS mass determination as described under Example 2.
• Synthetic ALB408-423 specifically blocks infection of X4-tropic HIV-I variants
• 5000 TZM-bl cells were seeded in 100 ⁇ l of DMEM (10% FCS, 100 U/ml of penicillin G and 100 ⁇ g/ml of streptomycin sulfate).
• ALB408-423 was dissolved in PBS (10 mg/ml).
• PBS 10 mg/ml
• 20 ⁇ l serial dilutions of ALB408-423 in PBS were added to cells and cells were subsequently infected with 0.5 ng of p24 antigen HIV-I in a total volume of 200 ⁇ l.
• HIV-I molecular clones differing in coreceptor tropism were used and generated as described (Papkalla et al., J. Virol. 76: 8455- 9, 2002).
• Figure 3 Chemically synthesized ALB408-423 specifically blocks X4 tropic HIV-I infection.
• TZM-bl cells containing indicated dilutions of peptide were infected with HIV-I variants differing in their coreceptor tropism. 3 days later GaI Screen assay was used to measure infection rates.
• CCR5 using) variant 92ht593.1 was blocked less efficiently.
• CCR5 tropic HIV-I variants were not inhibited even in the presence of very high doses of
• ALB408-423 (500 ⁇ g/ml) ( Figure 3B). Due to the specific inhibition of the infection caused by X4-tropic HIV-I variants, it is to be assumed that ALB408-423 interacts with the chemokine receptor CXCR4.
• ALB408-423 purified from hemofiltrate (Example 2) and the chemically synthesized ALB408-423 (Example 4) exhibited a dose-dependent inhibition of HIV-I replication in target cells providing evidence that ALB408-423 is a natural human HIV-I inhibitory molecule.
• Synthetic ALB408-423 dose dependently blocks CXCR4 tropic lentiviral infection.
• X4 tropic HIV-I NL4-3 and HIV-2ROD10 or CCR5 tropic HIV-I NL4-3 92thO14, HIV-1-7312 and SIVmac239 were generated by transient transfection of 293T cells and used to infect TZM-bl cells containing indicated concentrations of ALB408-423. Two days later infection rates were determined and calculated as described, (example 4). Results show that ALB408-423 dose dependently blocked X4 tropic HIV-I and HIV-2 infection (IC 50 ⁇ 10-20 ⁇ M) whereas the peptide had no effect on R5 tropic lentiviral infection demonstrating a specific inhibition of CXCR4 tropic HIV-I and HIV-2 (Fig. 4).
• FIG. 1 Antiviral activities of ALB derivatives.
• FIG. 9 Cytotoxicity assay of ALB derivatives. Serial dilutions of ALB derivatives were added to TZM-bl cells. After 2 days cellular ATP levels were measured using the CellTiter-Glo Luminescent Cell Viability Assay. Values were derived from triplicate measurements. % vitality rates were calculated relative to ATP levels in PBS (no peptide) containing cells (100 %).
• viruses differing in coreceptor use were generated by transfection of 293T cells with proviral plasmids (Papkalla et al., J. Virol. 76: 8455-9, 2002). Virus stocks were first titrated on TZM-bl cells. Then TZM-bl cells containing 100 ⁇ M of peptides were infected with infectivity normalized amounts of X4, dualtropic (X4/R5) or R5 tropic HIV-I.
• FIG. 10 ALB408-423 and truncated ALB derivatives specifically block X4 tropic HIV-I infection.
• TZM-bl cells either containing PBS or 100 ⁇ M of indicated peptides were infected with normalized infectivities of X4, dualtropic or R5 tropic HIV-I clones. Infection rates were measured 2 days post infection using the GalScreen assay. Shown are mean values (% of PBS treated control) ⁇ standard deviations derived from triplicate measurements.
• ALB408-423, ALB408-419 and ALB L408I-419 block X4 tropic HIV-I infection and replication in PBMC.
• peripheral blood mononuclear cells were isolated from Buffy coat derived from the DRK-Blutspendedienst Baden-W ⁇ rttemberg-Hessen using Ficoll density centrifugation. IxIO 6 PBMC per ml were stimulated with 1 ⁇ g/ml phytohemaggluti- nine (PHA, Oxoid, #3085280) and 10 ng/ml Interleukin 2 (IL-2, Strathmann, #9511192) for three days. Thereafter cells were pelleted and resuspended in IL-2 containing medium.
• PHA phytohemaggluti- nine
• IL-2 Interleukin 2
• FIG. 11 ALB408-423 and derivatives inhibit X4 tropic HIV-I infection of peripheral blood mononuclear cells (PBMC). Cells were incubated with indicated concen- trations of ALB408-423 or truncated variants and infected with X4 tropic HIV-I. Supernatants obtained after 6 days were analyzed by p24 ELISA. Shown are mean p24 antigen values (ng/ml) derived from triplicate infections ⁇ standard deviation.
• ALB 408-423 peptide inhibits binding of CXCL12 to CXCR4.
• CXCR4 In order to test the ability of ALB 408-423 to inhibit binding of the chemokine CXCL12 to its receptor, CXCR4, a fluorescent binding assay on whole living cells was performed as previously described (Valenzuela-Fernandez, et al.; 2001, JBC 276:26550-26558).
• the CXCR4 receptor is stably transfected in Human Embryonic Kidney (HEK) cells as a fusion protein with the EGFP fluorescent protein fused to the extracellular amino-terminal part of the receptor (EGFP-CXCR4).
• HEK Human Embryonic Kidney
• the human chemokines CXCL12 and CXCLl 2-TexasRed were synthesized as described (Amara et al., 1999, JBC 274:23916-23925; Valenzuela-Fernandez, et al., 2001, JBC 276:26550-26558).
• HEK293 cells expressing the fusion receptor, EGFP-hCXCR4 were harvested in phosphate-buffered saline supplemented with 5 mM EDTA, pH 7.4, centrifuged and resuspended in HEPES-bovine serum albumin buffer (10 mM HEPES, 137.5 mM NaCI, 1.25 mM MgCI2, 1.25 mM CaCI2, 6 mM KCI, 10 mM glucose, 0.4 mM NaH2PO4, 1% bovine serum albumin (w/v), pH 7.4) supplemented with protease inhibitors (40 ⁇ g/mL bestatin and bacitracin, 20 ⁇ g/mL phosphoramidon, 50 ⁇ g/mL chymostatin, and 1 ⁇ g/mL leupeptin).
• protease inhibitors 40 ⁇ g/mL bestatin and bacitracin, 20 ⁇ g/mL phosphoramidon, 50 ⁇ g
• the detected variation of fluorescence intensity can be quantified (Palanche et al., (2001), JBC 276:34853-34861; Vollmer et al., 1999, JBC 274: 37915-37922; Ilien et al., 2003, Neurochem 85:768-778) to derive binding constants of competitor.
• ALB408-423 dose dependency prevents interaction of CXCL12-Tr with its receptor CXCR4 (Fig. 12).
• ALB408-423 exhibits a dissociation constant (EC50) equal to 8 ⁇ 3 ⁇ M, corresponding to a KI value equal to 3 ⁇ 1 ⁇ M.
• the dissociation constant EC50 value is similar to the IC 5 O values obtained in HIV-I inhibition assays.
• ALB408-423 inhibits binding of CXCL12 to CXCR4.
• Real-time fluorescence monitoring of ligand-receptor interactions were performed using 293 cells expressing EGFP-hCXCR4. Cells were pre-incubated for 10 min in the absence or presence of various concentrations of ALB408-423. Then, CXCL12-TR (100 nM) was added and fluorescence was recorded until equilibrium was reached (300 sec).
• Peptide ALB408-423 does not induce Ca 2+ mobilization via CXC R4, CCR5 and CXCR4 and inhibits CXCL12-evoked calcium cellular responses
• the human chemokines CCL5 and CXCL8 were purchased from Becton Dickinson Biosciences (San Jose, CA). Ca 2+ mobilization assays using CXCR4, CCR5 or CXCRl expressing cells demonstrate that respective chemokine agonists CXCL12 (CXCR4), CCL5 (CCR5) and CXCL8 (CXCRl) induce Ca 2+ mobilization (Fig. 13) whereas ALB408-423 by itself does not induce any calcium response and hence does not exhibit CXCR4, CCR5 and CXCRl agonistic properties (Fig. 13).
• ALB408-423 is not a CXCR4, CCR5 or CXCRl agonist.
• HEK293 cells expressing indicated chemokine receptors were either treated with respective chemokines [10 nM CXCL12 (CXCR4); 20 nM CCL5 (CCR5) or 50 nM CXCL8 (CXCRl)] or 50 ⁇ M ALB408-423.
• Intracellular Ca 2+ responses were measured using a spectrofluorimeter. Fluorescence intensities obtained after treatment with ALB408-423 are shown relative to those measured for the respective chemokine (100 %).
• ALB408-423 specifically inhibits CXCL12-evoked Ca 2+ mobilization in CXCR4 expressing cells.
• Black bars respective chemokine only; grey bars: respective chemokine and 50 ⁇ M ALB408-423. Values shown are mean calcium peak responses from duplicate experiments relative to chemokine only treated cells (100%).
• ALB408-423 has CXCR4 antagonistic properties. Therefore, CXCR4 expressing cells were incubated with various concentrations of ALB408-423 and then treated with CXCL12. Ca 2+ responses were recorded. Data shown in Fig. 14A demonstrate that ALB408-423 inhibits CXCL12-evoked calcium responses in a dose-dependent manner and with an apparent inhibitory constant of 85 ⁇ g/ml. In order to gain insight into compound selectivity, we next characterized the effect of the peptide on calcium responses of various chemokine/receptor pairs. Consistent with data from Fig.
• ALB408-423 inhibits CXCLl 2-evoked CXCR4 internalization
• ALB 408-423 may also alter chemokine induced CXCR4 receptor internalization.
• EGFP-CXCR4 receptor expressing cells were split and grown for 2 days in 24-well plates on 12-mm glass coverslips coated with rat type I collagen.
• the cells were then incubated for periods ranging from 0 to 30 min in HEPES-BSA buffer supplemented with protease inhibitors containing either 100 nM CXCL12 or 50 ⁇ M of ALB408-423 or 100 nM CXCL12 plus 50 ⁇ M of ALB408-423 at 37 0 C. Internalization was stopped by placing cells on ice and washing them immediately with ice-cold HEPES-BSA buffer. The cells were then fixed in 4% paraformaldehyde in PBS for 15 min at 4 0 C and then incubated for 15 min in NH4CI 50 mM.
• Coverslips were mounted onto microscope slides using an anti- fading agent, M ⁇ viol (Calbiochem), maintained at room-temperature for 24 hours and then stored at -20 0 C. Cells were then analyzed with an inverted microscope (Leica) and a laser scanning confocal imaging system (Leica AOBS SP2 MP) using a HCX PL APO Ibd.BL 63X 1.40 OIL UV objective (n°506192). Electronic zoom was set to 3, the pinhole was 1 Airy, and the resulting pixel size was 0.154 ⁇ m.
• M ⁇ viol Calbiochem
• EGFP was excited with the 488 nm laser-line of the Argon laser and detected and amplified by one photomultiplier tube (PMT) in the so-called mCFP Channel from 495 to 550 nm (PMTl 610 High Voltage -HV-, offset 0). To obtain a good signal to noise ratio, the images were averaged from 4 consecutive acquisitions.
• PMT photomultiplier tube
• ALB408-423 blocks CXCL12 mediated CXCR4 internalization. Receptor endocytosis was monitored on HEK cells expressing EGFP-CXCR4 and analyzed by confocal microscopy immediately (0 min, upper panel) after addition of CXCL12, ALB408-423 or both compounds, or 30 min later (lower panel). After 30 min CXCL12 treated cells internalized CXCR4. In the presence of ALB408-423, CXCL12 mediated receptor internalization is abrogated.
• ALB408-423 blocks CXCL- 12 mediated migration of Jurkat T cells
• CXCL12-CXCR4 signaling plays a crucial role in several diseases such as HIV/AIDS, cancer, leukemia and arthritis.
• CXCL12 expressing organs, tissues or cells can attract CXCR4 expressing tumor cells and allow metastasis.
• migration assays were performed using Jurkat T cells expressing CXCR4 as model system (Princen et al., 2004, J.Virol. 78: 12996-13006).
• Jurkat T cells were suspended at 0.4 X 10 6 (200 ⁇ l) in medium containing 10% FBS, then the cell suspension (200 ⁇ l) was added to the upper compartment of 5 ⁇ m pore filter devices (Transwell, 24-well cell culture, Costar). Then, 600 ⁇ l culture medium with or without CXCL12 (100 ng/ml) was added to the lower compartment allowing attraction of cells from the upper compartment. To study inhibitory effects on CXCL12-induced Jurkat T cell migration, CXCL12 in the lower compartment was mixed with various concentrations of ALB 408-423. The cell culture plates were incubated for 2h in a cell culture incubator at 37°C.
• Fig. 16 demon- strate that ALB408-423 dose dependently inhibits CXCL12 mediated Jurkat T cell migration. At high concentrations (360 ⁇ g/ml) ALB408-423 blocked CXCL12 induced cell migration almost completely, comparable to rates observed in the absence of any peptide (no CXCL12, no ALB408-423). These data show that the CXCR4 antagonist ALB408-423 can inhibit attraction of tumour cells mediated by CXCLl 2.
• FIG. 16 ALB408-423 dose dependently blocks CXCL-12 mediated migration of Jurkat T cells.
• Jurkat T cells were added to the upper compartment of transwell devices with 5 ⁇ m pore filters. Then PBS, CXCR4 agonist CXCL12 (100 nM) or serial dilutions of ALB408-423 were added to the lower compartment of the cell culture plate. After 2 hrs incubation at 37°C, the number of migrated cells in the lower compartment was detected by measuring intracellular ATP levels using CellTiter-Glo® Luminescent Cell Viability Assay kit (Promega). All values represent mean numbers of migrated cells relative to CXCL12 only treated cells (100% migration) from a triplicate experiment ⁇ standard deviation.
• ALB408-423 binding to CXCR4 depends on the N-terminal amino-acid integrity.
• ALB409-423 is also inactive in the HIV-I inhibition assay indicating that the inability of ALB409-423 to bind CXCR4 also accounts for the loss of antiviral activity.
• all C terminal truncated ALB derivatives were still able to interact with the CXCR4 receptor (Fig. 17A) and blocked CXCL12 mediated Ca 2+ response (Fig. 17B) and exhibit a dissociation constant closed to the wild type peptide (30 ⁇ M), except for the smallest, 408-413, which has a lower affinity for the receptor (> 200 ⁇ M) and is also largely ineffective in blocking X4 tropic HIV-I infection (Fig. 6 and table 1).
• CXCR4 antagonists that are able to bind CXCR4 thereby preventing CXCL12 binding and signaling or X4 tropic HIV-I infection.
• FIG. 17 CXCR4 antagonistic activities of ALB derivatives.
• ALB derivatives abrogate CXCL12 binding to CXCR4.
• CXCR4 expressing HEK293 cells were treated with Texas Red labeled CXCL12 (CXCL12-Tr) in the presence or absence of ALB peptides.
• Real-time fluorescence monitoring of ligand-receptor interactions was carried out as described. Shown are bound CXCL12-Tr levels in the presence of ALB peptides relative to CXCL12-Tr only treated cells (100 % bound). Values were derived from a duplicate experiment.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• General Health & Medical Sciences (AREA)
• Medicinal Chemistry (AREA)
• Animal Behavior & Ethology (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Pharmacology & Pharmacy (AREA)
• General Chemical & Material Sciences (AREA)
• Public Health (AREA)
• Veterinary Medicine (AREA)
• Engineering & Computer Science (AREA)
• Virology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Oncology (AREA)
• Communicable Diseases (AREA)
• Molecular Biology (AREA)
• Pulmonology (AREA)
• Physical Education & Sports Medicine (AREA)
• Diabetes (AREA)
• Hematology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Genetics & Genomics (AREA)
• Biophysics (AREA)
• Biochemistry (AREA)
• Gastroenterology & Hepatology (AREA)
• Zoology (AREA)
• Toxicology (AREA)
• Biotechnology (AREA)
• Rheumatology (AREA)
• AIDS & HIV (AREA)
• Dermatology (AREA)
• Heart & Thoracic Surgery (AREA)
• Immunology (AREA)
• Neurology (AREA)
• Biomedical Technology (AREA)
• Tropical Medicine & Parasitology (AREA)
• Pain & Pain Management (AREA)

Priority Applications (10)

Applications Claiming Priority (2)

Publications (3)

Family

ID=39760841

Family Applications (1)

Country Status (12)

Cited By (9)

Families Citing this family (16)

Family Cites Families (6)

• 2007
  • 2007-07-03 DE DE102007030904A patent/DE102007030904A1/de not_active Withdrawn
• 2008
  • 2008-07-03 PT PT87746913T patent/PT2162462T/pt unknown
  • 2008-07-03 CA CA2691061A patent/CA2691061C/en active Active
  • 2008-07-03 ES ES17192177T patent/ES2770361T3/es active Active
  • 2008-07-03 DK DK17192177.8T patent/DK3275894T3/da active
  • 2008-07-03 ES ES08774691.3T patent/ES2655289T3/es active Active
  • 2008-07-03 WO PCT/EP2008/058566 patent/WO2009004054A2/en active Application Filing
  • 2008-07-03 HU HUE17192177A patent/HUE047454T2/hu unknown
  • 2008-07-03 US US12/452,480 patent/US9045563B2/en active Active
  • 2008-07-03 PT PT171921778T patent/PT3275894T/pt unknown
  • 2008-07-03 CN CN2008800233131A patent/CN101730709B/zh active Active
  • 2008-07-03 EP EP17192177.8A patent/EP3275894B1/en active Active
  • 2008-07-03 PL PL08774691T patent/PL2162462T3/pl unknown
  • 2008-07-03 CA CA2968054A patent/CA2968054C/en active Active
  • 2008-07-03 DK DK08774691.3T patent/DK2162462T3/da active
  • 2008-07-03 JP JP2010513986A patent/JP2010531655A/ja active Pending
  • 2008-07-03 EP EP08774691.3A patent/EP2162462B1/en active Active
  • 2008-07-03 HU HUE08774691A patent/HUE037931T2/hu unknown
  • 2008-07-03 PL PL17192177T patent/PL3275894T3/pl unknown
• 2014
  • 2014-06-18 JP JP2014125189A patent/JP5941497B2/ja active Active

Non-Patent Citations (3)

Also Published As

Similar Documents

Legal Events